Assessing the Impact of Acute and Chronic Exposure to Cigarette and Waterpipe Smoke Extracts on Telomeres, Telomerase, and Cancer Progression in a Lung Cancer Cell Line

Abstract

Introduction: Tobacco smoking imposes a worldwide health threat and is a major risk factor for many life-threatening diseases, which include cardiovascular and respiratory diseases, and many types of cancers, including lung cancer. Telomeres and telomerase are pivotal factors influencing cancer initiation and progression. In fact, in cancer cells, shortened telomeres contribute to genome instability and increased mutation rates, while telomerase, activated in more than 80% of cancer cells, maintains telomeres at a stable length, allowing the cells to gain an immortal phenotype and to proliferate indefinitely. Smoking on the other hand, is shown to contribute to telomere shortening in various types of human cells. Many studies have also shown that shorter telomeres and active telomerase in lung cancer are associated with poor patient prognosis, which includes a higher risk of mortality, recurrence, and a poor response to therapy. Aims: This study aims to assess the effects of acute and chronic exposure to cigarette (CSE) and waterpipe (WSE) smoke extracts on relative telomere length (RTL), telomerase expression and activity, and cancer progression in the A549 lung cancer cell line. Methods: MTT assay was used to estimate the IC20 and IC50 concentrations of WSE and CSE in A549 cells. Then, these concentrations were validated using trypan blue assay, cell cycle, and apoptosis. The cells were exposed for 24 hours to IC20 and IC50 concentrations, then for 12 weeks to IC20 concentrations of WSE and CSE. RTL and telomerase expression were measured at 24 hours and 6, 9, and 12 weeks, on isolated DNA by quantitative PCR (qPCR) and isolated cDNA by reverse transcriptase PCR (RT-PCR) respectively. Telomerase activity was measured using the telomerase repeated amplification protocol (TRAP) after protein isolation at 12 weeks. The migration ability of the cells was assessed using the migration assay and the expression of Epithelial to Mesenchymal Transition (EMT) markers including E-cadherin, Snail 1, Snail 2, and vimentin, was measured by RT-PCR. Results: The results of this study showed that there was a significant decrease in cell viability after 24 hours exposure to IC20 and IC50 of WSE and CSE. This was associated with a significant increase in the percentage of cells in Sub G0 phase for IC50 concentrations, and a significant increase of apoptosis for IC50 CSE. However, no cell cycle arrest was observed neither after 24 hours nor after 12 weeks of exposure. The qPCR results showed a significant decrease in RTL after 24 hours of exposure to 5 IC50 concentrations, as well as after 12 weeks of exposure to IC20 concentrations. Telomerase expression results showed significant decrease after 24 hours of exposure to both IC20 and IC50 concentrations for both extracts. However, after 9 weeks of exposure to IC20 concentrations, there was a significant increase in telomerase expression for waterpipe exposed cells, while a significant decrease was observed for cigarette exposed cells. Moreover, after 12 weeks of exposure, there was a significant increase in telomerase activity in waterpipe exposed cells compared to the control. The EMT expression results showed after 24 hours a significant increase in E-cadherin expression with IC50 WSE, and in Snail 2 expression with IC50 WSE and CSE. After 12 weeks of exposure to IC20 concentrations, there was a significant increase in Snail 1, Snail 2, and vimentin with CSE exposure, while no change was observed for WSE exposure. Finally, the migration assay showed no change in the migration ability of the cells neither after 24 hours nor after 12 weeks of exposure. Conclusion: This study is the first to investigate the in vitro effects of both acute and chronic exposure to cigarette and waterpipe smoke extracts on telomeres and telomerase. The results indicate that both acute and chronic exposure to both extracts lead to significant telomere shortening. It also shows that while acute exposure results in a significant decrease in telomerase expression for both extracts, chronic exposure leads to increased telomerase activity, which was only significant for waterpipe. Moreover, the study found that chronic exposure to CSE may induce epithelial to mesenchymal transition. However, neither acute nor chronic exposure affected the cells' migration ability.